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Hydrobiologia 479: 75–81, 2002. 75 © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Growth and physiological responses of candel and gymnorrhiza to livestock wastewater

Y. Ye 1,2 &NoraF.Y.Tam2 1Marine Biotechnology Key Lab, Ningbo University, Ningbo, Zhejiang, China E-mail: [email protected] 2Department of Biology and Chemistry, City University of Hong Kong, Hong Kong

Received 15 March 2001; in revised form 6 March 2002; accepted 31 March 2002

Key words: Kandelia candel, Bruguiera gymnorrhiza, , livestock wastewater, salinity

Abstract Growth and physiological responses of two mangrove species (Kandelia candel and Bruguiera gymnorrhiza)to livestock wastewater under two salinity conditions (seawater with salinity of 30þ and freshwater) were examined in greenhouse pot-cultivation systems for 144 days. Wastewater treatment significantly enhanced growth of Kandelia candel and Bruguiera gymnorrhiza in terms of stem height, stem basal diameter, leaf production, maximum unit leaf area and relative growth rate. Wastewater discharges and salinity levels did not significantly change biomass partitioning of Kandelia candel, however, more biomass of Bruguiera gymnorrhiza was allocated to leaf due to wastewater discharges. In Bruguiera gymnorrhiza, contents of chlorophyll a and chlorophyll b increased with wastewater discharges but such increase was not observed in Kandelia candel. On the other hand, livestock wastewater increased leaf electric conductance in Kandelia candel but not in Bruguiera gymnorrhiza. The per- oxidase activity in stem and root of Kandelia candel under both salinity conditions increased due to wastewater discharges, while the activity in root of the treated Bruguiera gymnorrhiza seedlings decreased under freshwater condition but increased at seawater salinity. The superoxide dismutase activity in treated Bruguiera gymnorrhiza decreased but did not show any significant change in Kandelia candel receiving livestock wastewater.

Introduction removal should be selected. The species should also be tolerant to wastewater stress. So growth and physiolo- Natural and constructed wetlands including man- gical responses of the wastewater treated should groves have been considered to be low-cost and effect- be an important consideration when selecting the ap- ive wastewater treatment systems and are especially propriate species for wastewater treatment. efficient in nutrient removal (Clough et al., 1983; Cor- are naturally distributed in areas with a bitt & Bowen, 1994; Breaux et al., 1995). Nutrients, wide range of water salinity, from nearly freshwater especially nitrogen and phosphorus are often deficient to seawater (Lin, 1999). Habitat salinity may influ- in coastal inter-tidal areas such as mangrove wetlands ence the N uptake by mangrove plants, and affect (Boto & Wellington, 1983; Clough et al., 1983; Lin, their growth and physiological responses to wastewa- 1999). Therefore, mangrove wetlands are particularly ter addition. Naidoo (1990) suggested that growth suitable for the treatment of wastewater rich in nu- enhancement of Bruguiera gymnorrhiza in response to trients. In a mangrove ecosystem, mangrove is N addition occurs only at a low salinity. The present the most important component. If mangroves were investigation therefore aims to compare growth and to be used as the treatment system for nutrient-rich physiological responses of two dominant mangrove wastewater such as livestock sewage, suitable man- plant species in Hong Kong SAR and South-east Asia, grove plant species that is most efficient in nutrient Bruguiera gymnorrhiza and Kandelia candel,tolive- 76

Table 1. Characteristics of livestock wastewater, tap water and seawater used in this experiment

Parameter Wastewater Tap water Seawater

pH 7.24 (7.00∼7.35) 7.64 (7.10∼8.03) 8.01 (7.32∼8.26) − Conductivity (ms cm 1) 1.39 (1.14∼1.77) 0.22 (0.18∼0.23) 36.60 (34.83∼38.49) − TOC (mg l 1) 65.9 (48.1∼88.3) 2.3 (0.5∼5.4) 5.2 (2.1∼7.0) + −1 NH4 -N (mg l ) 36.1 (21.2∼58.7) 0.3 (0.0∼1.0) 1.9(0.0∼5.9) − −1 NOx -N (mg l ) <0.05 <0.05 <0.05 3− −1 PO4 -P (mg l ) 53.7 (22.5∼94.6) 0.2 (0.0∼0.5) 0.2(0.0∼0.7)

Mean and range (in brackets) values of 9 samples are shown. stock wastewater. The effect of salinity on these re- 0.64 ± 0.05 cm and the stem height was 25.2 ± 3.9 sponses was also evaluated. This work is of significant cm (n=36). importance to the selection of the most suitable plant During the treatment period, each pot was daily species of mangrove wetlands to treat wastewater rich watered with 300 ml treatment liquid. For each plant in nutrients. species, the following four treatments, each in triplic- ate and lasted for 144 days, were set up: (1) F, each pot was irrigated with tap water every day, (2) FW, each pot was irrigated with wastewater in Materials and methods every three days and tap water in every other day, (3) S, each pot was irrigated with artificial seawater Experimental design (salinity of 30þ, prepared by dissolving a commer- cial salt purchased from Instant Ocean, Aquarium In April 1997, mature propagules of Bruguiera gym- Systems, Inc., Mentor, Ohio) every day, norrhiza were collected from Mai Po Mangrove ◦  ◦  (4) SW, each pot was irrigated with wastewater in Nature Reserve (114 05 E, 22 32 N) in Hong every three days and seawater in every other day. Kong SAR and planted into plastic pots (18 cm in diameter and 20 cm in height), with four individuals Growth and biomass partitioning analysis per pot, in a greenhouse. In November 1998, 1.5 years old Kandelia candel seedlings were transplanted from At the beginning and at the end of the experiment, the Wong Chuk Wan mangrove swamp of Hong Kong into stem basal diameter (D) at the first stem node and the greenhouse pots with three individuals per pot. Each stem height (H) (excluding hypocotyl) of each plant pot contained 4 kg soils freshly collected from Sai were measured. At the end of the experiment, biomass Keng mangrove swamp, HKSAR. The soil is a loamy- partitioning was determined by 105 ◦C dry weights sandy soil with 73.11%, 15.93% and 10.96% of sand, of shoot (leaf and stem) and root of each individual silt and clay particles, respectively. Each pot was daily plant. Hypocotyl was excluded in either shoot or root irrigated with 300 ml tap water and excess water was biomass measurement, mainly because the weight of able to drain freely from the six draining holes (0.6 cm hypocotyl did not change during the experiment. Data in diameter) at the bottom by gravitational force. on total biomass per pot (B), average stem basal dia- On 3 March 1999, 12 pots of B. gymnorrhiza and meter per pot (D) and average stem height per pot 12 pots of K. candel were used for treating livestock (H) collected at the end of the experiment were used wastewater. The livestock sewage was collected from to fit the non-destructive allometric equations sugges- Ta Kwu Ling Pig Farm of Hong Kong SAR after be- ted by Snedaker & Snedaker (1984). The calculated ing primarily treated by sedimentation and secondarily equations for the two species were as follows: treated by aeration. The wastewater had high P content + 3− B. gymnorrhiza : log B = 0.9998 + and low NH4 -N/PO4 –P ratio of 1:1.5 (Table 1). The stem basal diameter and stem height of Bruguiera 0.5416 log (D2H)(n = 12, p < 0.01) gymnorrhiza at the start of the experiment were 0.82 = + ± 0.08 cm and 24.2 ± 3.2 cm, respectively (n=48), K. candel:logB 0.6108 0.6450 while the basal stem diameter of Kandelia candel was log (D2H)(n = 12,p <0.01) 77

The initial biomass per pot was then estimated accord- ing to the equations. The relative growth rate (RGR) was calculated as

ln B –lnB RGR = 2 1 . t2 − t1

B1 and B2 were total biomass at the beginning (t1)and at the end (t2) of the experimental period (Hunt, 1978).

Physiological analysis

About 0.1 g fresh tissues of the third pair of leaves from the top were ground in cold mortar with 10 ml 80% acetone. The homogenate was centrifuged at 10 000× g for 3 min. The contents of chlorophyll a, chlorophyll b, total chlorophyll and total carotenoid were determined according to Lichtenthaler & Well- burn’s method (1983). The methods used to determine leaf electric conductance and root activity were the same as described by Zhang (1990). The methods for extraction and determination of peroxidase (POX) and superoxide dismutase (SOD) activity were similar to those described by Liu & Zhang (1994) with minor modifications. Portions of fresh plant materials were ground and homogenized with five times of 62.5 mmol l−1 phosphate buffer (pH 7.8, including 0.4% polyvinyl pyrrolidone) in an ice bath. The homogenate was centrifuged at 1500 × g for 20 min. The supernatant, the extract of POX and SOD, was stored at 4 ◦C before the enzyme activity was determined. Figure 1. Growth of Kandelia candel (Kc) and Bruguiera gymnor- For the POX activity assay, 20 µl of the enzyme rhiza (Bg) at the end of 144 days treatment (for each species, means extract was diluted to 1 ml with deionized water, with different letters are significantly different at p<0.05 level). mixed with 3 ml combined reagent (consists of 500 −1 ml 0.1 mol l phosphate buffer at pH 7.0, 280 µl was measured. An enzyme unit was calculated as 50% guaiacol and 190 µl hydrogen peroxide). The ab- inhibition of the maximum photo-reduction of NBT. sorbance at 470 nm was measured at every minute interval. An increase of 0.01 absorbance per minute Data analysis was considered as one unit of POX activity. The SOD activity was determined by the degree of inhibition Mean and standard deviation values of triplicates were to photo-reduction of nitro blue tetrazolium (NBT). calculated. A parametric two-way analysis of variance The following reagents in a sequence of 2.4 ml 62.5 (ANOVA) test was employed to examine any signific- mmol l−1 phosphate buffer, 0.2 ml 0.06 mmol l−1 ant difference among wastewater treatments, between riboflavin, 0.2 ml 30 mmol l−1 methionine, 0.1 ml salinity conditions, and interactions between wastewa- −1 0.003 mmol l Na2EDTA, 20 µl enzyme extract, and ter and salinity. The Student–Newman–Keuls multiple 0.2 ml 1.125 mmol l−1 NBT were mixed. In a blank comparison method was used if significant difference set up to determine the maximum photo-reduction of was found among treatments. NBT, the enzyme extract was substituted by the buffer solution. The reaction was carried out under illumina- tion (400 lux) for 1 h, and the absorbance at 560 nm 78

Table 2. Results of two-way ANOVA tests showing the effects of livestock wastewater treatments and salinity conditions on the growth (cumulative increase in stem height: H; cumulative in- crease in stem basal diameter: D; cumulative increase in leaf number production: L; maximum unit leaf area: MLA; relative growth rate: RGR) and biomass partitioning (root weight ratio: RWR; stem weight ratio: SWR; leaf weight ratio: LWR; biomass ratio of root – shoot: R/S) of Kandelia candel (Kc) and Bruguiera gymnorrhiza (Bg)

Source of variance H D L MLA RGR RWR SWR LWR R/S

Kc Salinity (S) NS ∗ ∗∗∗ NS NS NS NS NS NS ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ Wastewater (W) NS NS NS NS S×W NSNSNSNSNSNSNSNSNS

Bg ∗∗∗ ∗ ∗∗ Salinity (S) NS NS NS NS NS NS Wastewater (W) ∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗ ∗∗∗ NS ∗∗∗ ∗∗∗ S×WNS∗ NS NS NS NS NS NS NS ∗ ∗∗ Note: NS (not significant), (p<0.05), (0.05

Results and discussion

Growth responses to livestock wastewater

Discharges of livestock wastewater significantly en- Figure 2. Biomass partitioning in terms of root weight ratio to hanced all growth parameters, including stem height, whole biomass (RWR); stem weight ratio to whole biomass (SWR); stem basal diameter, leaf production, maximum unit leaf weight ratio to whole biomass (LWR); biomass ratio of root – leaf area and relative growth rate of Kandelia candel shoot (R/S) of Kandelia candel (Kc) and Bruguiera gymnorrhiza and Bruguiera gymnorrhiza seedlings (Fig. 1, Table (Bg) at the end of 144 days treatment (for each species, means with different letters are significantly different at p<0.05 level). 2). For the controls, the two salinity levels had no significant effects on the increases of stem height and relative growth rates of both species. For wastewater treatments, Bruguiera gymnorrhiza seedlings had sig- Table 3. Results of two-way ANOVA showing the effects of live- nificantly more increases in stem diameter and leaf stock wastewater treatments and salinity conditions on leaf pigments, area than Kandelia candel seedlings under both sa- including chlorophyll a (Chl a), chlorophyll b (Chl b), total chloro- linity conditions. McKee (1995) also indicated that phyll (Chl), ratio of chlorophyll a to chlorophyll b (a/b), total higher nutrient levels resulted in greater investment in carotenoid (Car), leaf electric conductance (LC), leaf water con- tent (LWC), and root activity (RA) of Kandelia candel (Kc) and leaf area and leaf production of seedlings of three man- Bruguiera gymnorrhiza (Bg) grove species, namely Rhizophora mangle, Avicennia germinans and Laguncularia racemosa. Source of variance Chl a Chl b Chla/b Car LC LC RA In general, Kandelia candel seedlings had higher Kc RWR (root weight – total biomass ratio), SWR (stem Salinity (S) NS NS NS ∗∗∗ NS ∗∗∗ NS ∗ ∗∗∗ ∗∗∗ ∗∗ weight – total biomass ratio) and R/S ratio (root – Wastewater (W) NS NS NS NS NS × ∗∗∗ ∗ shoot biomass ratio) but lower LWR (leaf weight to S WNSNSNS NS NS NS total biomass ratio) than Bruguiera gymnorrhiza seed- Bg ∗ ∗∗ ∗∗∗ ∗ lings (Fig. 2). Salinity levels and livestock wastewater Salinity (S) NS NS NS NS treatments did not significantly change biomass parti- Wastewater (W) ∗∗∗ ∗∗∗ ∗∗∗ NS ∗∗∗ NS ∗∗∗ NS × ∗∗∗ tioning of Kandelia candel (Table 2). On the contrary, S W NS NS NS NS NS ∗ ∗∗ Bruguiera gymnorrhiza seedlings receiving livestock Note: NS (not significant), (p<0.05), (0.05

Figure 4. Leaf electric conductance and leaf water content of Kan- delia candel (Kc) and Bruguiera gymnorrhiza (Bg) at the end of 144 days treatment (for each species, means with different letters are significantly different at p<0.05 level).

oids (pigments protect chlorophyll from oxidation) and the ratio of chlorophyll a to chlorophyll b in leaf of Kandelia candel seedlings treated with livestock wastewater increased dramatically under freshwater condition but such increase was not found under sea- water condition. Unlike Kandelia candel, Bruguiera gymnorrhiza treated with wastewater had significantly higher contents of leaf chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids than the con- trols under both freshwater and seawater conditions. Significant interactions between salinity levels and wastewater treatments were found in chlorophyll con- tents of Bruguiera gymnorrhiza but not in Kandelia Figure 3. Leaf pigments, including chlorophyll a (Chl a), chloro- candel. phyll b (Chl b), total chlorophyll (Chl), ratio of chlorophyll a to The two species responded differently to livestock chlorophyll b (a/b), and total carotenoid (Car) of Kandelia candel (Kc) and Bruguiera gymnorrhiza (Bg) at the end of 144 days treat- wastewater treatments in leaf electric conductance ment (for each species, means with different letters are significantly (Fig. 4 and Table 3). The electric conductance of different at p<0.05 level). Kandelia candel seedlings treated with wastewater was significantly higher than their respective con- trols. Similarly, Chen et al. (1995) and Wong et al. ratio but a significant increase in LWR, suggesting that (1997) also reported that Kandelia candel and Aegi- more biomass was allocated to leaf. ceras corniculatum seedlings treated with synthetic wastewater had higher electric conductance values. Physiological responses However, there was no significant increase in electric conductance in wastewater treated Bruguiera gymnor- Contents of Chlorophyll a, chlorophyll b, and total rhiza under freshwater condition and the value even chlorophyll in leaves of Kandelia candel treated with dropped under seawater condition. These results in- livestock wastewater were not significantly different dicated that Bruguiera gymnorrhiza was more tolerant from those found in the controls (Fig. 3 and Table 3). to livestock wastewater than Kandelia candel. This is similar to the results of Aegiceras corniculatum Similar to that reported by Chen et al. (1995) on ef- and Kandelia candel receiving synthetic sewage under fects of artificial wastewater on water contents of man- simulated tidal conditions (Chen et al., 1995; Wong grove seedlings, the present study also showed that et al., 1997). However, the content of total caroten- livestock wastewater did not significantly change leaf 80

Table 4. Results of two-way ANOVA showing the effects of live- stock wastewater treatments and salinity conditions on activities of oxidation-resistant enzymes, including peroxidase (POX) and su- peroxide dismutase (SOD) of Kandelia candel (Kc) and Bruguiera gymnorrhiza (Bg)

Source of variance POX SOD Leaf Stem Root Leaf Stem Root

Kc Salinity (S) ∗ ∗∗∗ Figure 5. Root activity (µg α-naphthylamine per gram of fresh Wastewater (W) NS NS ∗∗ ∗∗ ∗ weight per hour) of Kandelia candel (Kc) and Bruguiera gymnor- S×WNS NS NS rhiza (Bg) at the end of 144 days treatment (for each species, means with different letters are significantly different at p<0.05 level). Bg NS NS NS NS NS NS Salinity (S) ∗∗∗ ∗ ∗∗∗ ∗ Wastewater (W) NS NS ∗∗∗ ∗ ∗∗∗ S×WNSNSNS ∗∗∗ ∗ NS NS NS NS ∗ ∗∗ Note: NS (not significant), (p<0.05), (0.05

Figure 6. Peroxidase (POX) activity (×103 unit per gram of fresh weight) in leaf, stem and root of Kandelia candel (Kc) and Bruguiera gymnorrhiza (Bg) at the end of 144 days treatment (for each species, means with different letters are significantly different at p<0.05 level).

Figure 7. Superoxide dismutase (SOD) activity (unit per gram of fresh weight) in leaf, stem and root of Kandelia candel (Kc) and water contents of Kandelia candel (Fig. 4 and Table Bruguiera gymnorrhiza (Bg) at the end of 144 days treatment (for each species, means with different letters are significantly different 3). However, water contents in leaf of Bruguiera gym- at p<0.05 level). norrhiza seedlings treated with wastewater increased significantly under both freshwater and seawater con- ditions. It has been proposed that leaf water contents The discharge of livestock wastewater did not sig- could reflect a plant’s activity, and a higher value nificantly change the root activity of both Kandelia indicated the plant possessed a high metabolic rate candel and Bruguiera gymnorrhiza (Fig. 5 and Table and a rapid growth (Chen et al., 1995). Therefore, in 3), similar to the result obtained by Chen et al. (1995) terms of water content, both species were tolerant to who examined the responses of Kandelia candel to livestock wastewater although Bruguiera gymnorrhiza artificial wastewater. This also indicates that livestock appeared to be more tolerant than Kandelia candel. wastewater did not damage root systems of Kandelia 81 candel and Bruguiera gymnorrhiza, and both species References might be somewhat tolerant to sewage discharge. Due to wastewater addition, the POX activity in Boto, K. G. & J. T. Wellington, 1983. Nitrogen and phosphorus stem and root of Kandelia candel increased under sea- nutritional status of a northern Australian mangrove forest. Mar. Ecol. Prog. Ser. 11: 63–69. water condition but the changes in treated leaf were Breaux, A. S., S. Farber & J. Day, 1995. Using natural coastal insignificant (Fig. 6 and Table 4). Chen et al. (1995) wetlands systems for wastewater treatment: an economic benefit also observed that leaf POX activity did not signifi- analysis. J. 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